The saliva of the blood-sucking insect, Rhodnius prolixus, originally from the Amazon river basin, which carries the parasite Trypanasoma cruzi, the vector of Chagas' disease, contains a suite of heme proteins that we have named Nitrophorins (NPs). The NPs act as vasodilators, anti-platelet aggregation and anti-histaminic agents when injected into the victim. These properties aid the insect in obtaining an ample blood meal, and thus make more likely the transmission of the trypanosome from the insect feces to humans. The trypanasome multiplies intracellularly and is released upon cell death to enter the blood stream and infect distant cells. Chagas' disease slowly weakens the autonomic muscles of the heart, lungs and/or gut and thereby may cause death some 30 or more years later. There is no cure. The North American blood supply is in danger of contamination by the trypanosome. The NPs are novel ferriheme proteins that are reversible carriers of nitric oxide (NO) and tight binders of histamine. They are the only known heme proteins with the heme inside a ?-barrel (lipocalin fold). Our hypothesis is that extreme ruffling of the heme (revealed by high-resolution X-ray crystal structures) is the key to their unusual properties, including stabilization of FeIII and reversible NO binding. Specific aims for the next grant period are: 1) To investigate NP4 and the native terminus-containing constructs of NP1, NP2 and NP3 or their X1A mutants, in which the recombinantly-produced proteins' Met0s are cleaved. The thermodynamics and kinetics of their function, and the roles that individual NP proteins play in making a suite of closely related proteins such a successful strategy for a blood-sucking insect will be studied. 2) To investigate the ps to min dynamics of apo- and holo-NP2 and NP4 in order to understand the fast and slow timescale dynamics of these ?-barrel proteins, how the plasticity of the ?-barrel is affected by adding the prosthetic group and then NO or histamine to the apoprotein, and how the dynamics of the two proteins differ. The slow (5s to min) dynamics of NP2-ligand complexes will be compared to those of NP4 to understand why the rates of NO, but not histamine release are a factor of 40 slower for NP2-D1A than for NP4, and how the dynamics of loop opening and closing are able to control the rate of ligand binding and release. 3) To investigate, by measuring Eos of additional charge mutants and by theoretical calculations, why some buried and surface charge mutants of NP2-D1A affect the Eo of the heme more strongly than others at the same distance from iron. The many charged residues of the NPs make them a useful laboratory for probing the effects of charges on the Eo of Fe. Negative charges stabilize the FeIII form of the protein, which is able to release NO with nM Kds, while the FeII form has fM Kds and thus binds NO irreversibly, preventing vasodilation. We envision that the information obtained in this work will allow understanding the roles of the N-terminus, the dynamics of the proteins and their response to both heme and ligand binding, and the roles of the charged residues of the NPs in protein function. These findings will be broadly applicable to other heme proteins and to metalloproteins in general. PUBLIC HEALTH RELEVANCE: The saliva of the blood-sucking insect, Rhodnius prolixus, originally from the Amazon river basin, which carries the parasite Trypanasoma cruzi, the vector of Chagas' disease, contains a suite of heme proteins that we have named Nitrophorins (NPs). The NPs act as vasodilators, anti-platelet aggregation and anti-histaminic agents when injected into the victim. These properties aid the insect in obtaining an ample blood meal, and thus make more likely the transmission of the trypanosome from the insect feces to humans. The information obtained in the work proposed will allow understanding the roles of the N-terminus of the nitrophorins, the dynamics of these proteins and their response to both heme and ligand binding, and the roles of the charged protein residues of the NPs in protein function.